Abstract
Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what ...extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.
Exosomes are small membrane vesicles (50–90 nm in diameter) secreted by most hematopoietic cells. We provide here the first evidence for the presence of exosomes in vivo, in the blood. Plasma samples ...of all healthy donors tested (n = 15) contain vesicles that are similar in shape, size and density to the previously described exosomes. They were clearly identified by electron microscopy after isolation by differential ultracentrifugation or immunoisolation with CD63-coated latex beads. We performed their biochemical characterization by western blot analysis and by flow cytometry after vesicle adsorption onto latex beads using a panel of mAbs. We observed that these plasma-derived vesicles contain tetraspanin molecules such as CD63, CD9, CD81 as well as class I and class II MHC molecules and Lamp-2 (i.e. proteins that are known to be enriched in exosomes). In addition, these vesicles float on sucrose gradient at a density similar to exosomes. Our results demonstrate that blood is a physiological fluid for exosome circulation in the body, suggesting their role in cell–cell or organ–organ communications as carriers for molecules that need to reach distant cell targets.
The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is ...unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space.
An important channel of cell-to-cell communication is direct contact. The immune synapse is a paradigmatic example of such type of interaction: it forms upon engagement of antigen receptors in ...lymphocytes by antigen-presenting cells and allows the local exchange of molecules and information. Although mechanics has been shown to play an important role in this process, how forces organize and impact on synapse function is unknown. We find that mechanical forces are spatio-temporally patterned at the immune synapse: global pulsatile myosin II-driven tangential forces are observed at the synapse periphery while localised forces generated by invadosome-like F-actin protrusions are detected at its centre. Noticeably, we observe that these force-producing actin protrusions constitute the main site of antigen extraction and endocytosis and require myosin II contractility to form. The interplay between global and local forces dictated by the organization of the actomyosin cytoskeleton therefore controls endocytosis at the immune synapse.
Engagement of the B cell receptor (BCR) by surface-tethered antigens (Ag) leads to formation of a synapse that promotes Ag uptake for presentation onto major histocompatibility complex class II ...(MHCII) molecules. We have highlighted the membrane trafficking events and associated molecular mechanisms involved in Ag extraction and processing at the B cell synapse. MHCII-containing lysosomes are recruited to the synapse where they locally undergo exocytosis, allowing synapse acidification and the extracellular release of hydrolases that promote the extraction of the immobilized Ag. Lysosome recruitment and secretion results from the polarization of the microtubule-organizing center (MTOC), which relies on the cell division cycle (Cdc42)-downstream effector, atypical protein kinase C (aPKCζ). aPKCζ is phosphorylated upon BCR engagement, associates to lysosomal vesicles, and is required for their polarized secretion at the B cell synapse. Regulation of B lymphocyte polarity therefore emerges as a central mechanism that couples Ag extraction to Ag processing and presentation.
► Lysosomes are secreted at the B cell immune synapse in response to MTOC polarization ► MTOC polarization and lysosome secretion rely on Cdc42 and its effector protein aPKCζ ► Lysosome secretion results in synapse acidification and local release of hydrolases ► Secretion of lysosomal proteases couples antigen extraction to antigen processing
Dendritic cells (DCs) sample peripheral tissues of the body in search of antigens to present to T cells. This requires two processes, antigen processing and cell motility, originally thought to occur ...independently. We found that the major histocompatibility complex II-associated invariant chain (Ii or CD74), a known regulator of antigen processing, negatively regulates DC motility in vivo. By using microfabricated channels to mimic the confined environment of peripheral tissues, we found that wild-type DCs alternate between high and low motility, whereas Ii-deficient cells moved in a faster and more uniform manner. The regulation of cell motility by Ii depended on the actin-based motor protein myosin II. Coupling antigen processing and cell motility may enable DCs to more efficiently detect and process antigens within a defined space.
Complete activation of B cells relies on their capacity to extract tethered antigens from immune synapses by either exerting mechanical forces or promoting their proteolytic degradation through ...lysosome secretion. Whether antigen extraction can also be tuned by local cues originating from the lymphoid microenvironment has not been investigated. We here show that the expression of Galectin-8—a glycan-binding protein found in the extracellular milieu, which regulates interactions between cells and matrix proteins—is increased within lymph nodes under inflammatory conditions where it enhances B cell arrest phases upon antigen recognition in vivo and promotes synapse formation during BCR recognition of immobilized antigens. Galectin-8 triggers a faster recruitment and secretion of lysosomes toward the B cell-antigen contact site, resulting in efficient extraction of immobilized antigens through a proteolytic mechanism. Thus, extracellular cues can determine how B cells sense and extract tethered antigens and thereby tune B cell responses in vivo.
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•Galectin-8 reinforces B cell arrest phases upon antigen recognition in vivo•Galectin-8 sustains BCR signaling during recognition of immobilized antigens•This enhances lysosome secretion and favors the proteolytic extraction of antigens•Galectin-8 improves the capacity of B cells to present antigens to helper T cells
Obino et al. report that Galectin-8 interacts with the BCR, promotes B cell arrest phases during surface-tethered antigen encounter, and facilitates synapse formation and lysosome secretion, which favors the proteolytic extraction of antigens. Consequently, Galectin-8 increases the capacity of B cells to present antigens to helper T cells in vivo.
Cell polarity is an essential and highly conserved process governing cell function. Cell polarization is generally triggered by an external signal that induces the relocation of the centrosome, thus ...defining the polarity axis of the cell. Here, we took advantage of B cells as a model to study cell polarity and perform a medium-throughput siRNA-based imaging screen to identify new molecular regulators of polarization. We first identified candidates based on a quantitative proteomic analysis of proteins differentially associated with the centrosome of resting non-polarized and stimulated polarized B cells. We then targeted 233 candidates in a siRNA screen and identified hits regulating the polarization of the centrosome and/or lysosomes in B cells upon stimulation. Our dataset of proteomics, images, and polarity indexes provides a valuable source of information for a broad community of scientists interested in the molecular mechanisms regulating cell polarity.
Exosomes are small vesicles secreted by different immune cells and which display anti‐tumoral properties. Stimulation of RBL‐2H3 cells with ionomycin triggered phospholipase D2 (PLD2) translocation ...from plasma membrane to intracellular compartments and the release of exosomes. Although exosomes carry the two isoforms of PLD, PLD2 was enriched and specifically sorted on exosomes when overexpressed in cells. PLD activity present on exosomes was clearly increased following PLD2 overexpression. PLD2 activity in cells was correlated to the amount of exosome released, as measured by FACS. Therefore, the present work indicates that exosomes can vehicle signaling enzymes.
Exosomes are small vesicles (60–100 nm) secreted by various cell types upon the fusion of endosomal compartments with the plasma membrane. Exosomes from antigen‐presenting cells (APC), such as B ...lymphocytes and dendritic cells (DC), bear MHC class II molecules. In addition, the injection of DC‐derived exosomes was reported to elicit potent T cell responses in vivo. Here, we analyzed the activation of specific T cells by MHC class II‐bearing exosomes in vitro. The rat mast cell line, RBL‐2H3, was engineered to express human class II molecules uniformly loaded with an antigenic peptide HLA‐DR1–hemagglutinin (HA). These cells secreted exosomes bearing DR1 class II molecules upon stimulation by a calcium ionophore or IgE receptor cross‐linking. Exosomes bearing DR1–HA(306–318) complexes activated HA/DR1‐specific T cells only weakly, whereas the cross‐linking of such exosomes to latex beads increased stimulation of specific T cells. By contrast, the incubation of free exosomes with DC resulted in the highly efficient stimulation of specific T cells. Thus, exosomes bearing MHC class II complexes must be taken up by professional APC for efficient T cell activation.
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